to the Luer-Lok@ extension of the Minicolumn. DNA purification resin (7 M guanidine

HC1), 1 mL, was pipetted in the barrel, followed by the cell lysate. The syringe plunger

was inserted in the barrel and used to push the slurry through the Minicolumn. The

syringe was detached from the Minicolumn and the plunger removed from the syringe

barrel. The barrel was then reattached to the Minicolumn. Column wash solution (80 mM

potassium acetate, 8.3 mM Tris-HCI (pH 7.5), 40 C1M EDTA, 55% ethanol), 2 mL, were

pipetted into the barrel of the Minicolumn/syringe assembly, and the solution was pushed

through the Minicolumn by the plunger. The syringe was removed, and the Minicolumn

was transferred to a 1.5 mL microcentrifuge tube, which was centrifuged at 10,000g for 2

min to dry the resin. The Minicolumn was transferred to a new 1.5 mL microcentrifuge

tube and 50 CIL nuclease-free water was added to the column and left for 1 minute. The

DNA was eluted by centrifuging at 10,000g for 20 sec. The Minicolumn was removed

and discarded, and the DNA solution stored at -200C. Products were visualized by 1%

agarose gel electrophoresis run at 100V, using 3 CIL of purified DNA and 10 CIL

quantitative 1kb plus DNA ladder (0.5 Gig).

 Digestion with ecoRI. To ensure that the two DNA bands seen in the purified

plasmid DNA run on agarose gel were due to supercoiling of the DNA and not

contamination, the plasmid DNA was digested with the restriction enzyme ecoRI (pGEM

T-Easy Vector has restriction sites on either side of the insert). Plasmid DNA, 3 CIL, was

added to a tube containing 0.2 CIL acetylated BSA, 2 CIL 10x buffer, and 14.3 CIL DNase-

free water, and mixed by pipetting. ecoRI restriction enzyme (12 U/CIL), 0.5 CIL, was

added and the solution mixed by pipetting. The tubes were briefly centrifuged and